Healing wounds in human skins is a major medical problem, particularly in the elderly patient population. According to the Wound Healing Society, about 15% of older adults suffer from chronic, hard-to-heal wounds [1]. It is also estimated that about 18% of diabetic patients over the age of 65 years will have chronic, nonhealing skin ulcers [2]. To improve the healing process, researchers have been considering topically applying epidermal growth factor as a promising therapy. This therapy has been shown to accelerate wound closure of acute wounds in patients [3-5]. However, due to the high cost and other practical considerations, this strategy has not been commercially viable as a general solution for wound healing. So far, only platelet-derived growth factor has been approved by the Federal Drug Administration for treatment of nonhealing diabetic foot ulcers. Even with this therapy, practitioners have found it to be limiting and not always successful. One difficulty associated with the topical application of growth factors is that the wound bed is often laden with proteolytic enzymes which tend to degrade and nullify the applied agent.
To investigate better approaches of skin wound healing, various models of skin diseases have been used. In particular, a genetically inherited skin disease known as Dystrophic forms of Epidermolysis Bullosa (DEB) in children has provided valuable insights.
DEB is an incurable genetic disease caused by a gene defect in the gene that encodes for type VII collagen. Children who suffer from DEB are born with skin fragility, blistering, and repeated wounding and healing of their skin wounds [6]. In these children, their wounds will typically heal with fibrosis, scarring, and small epidermal inclusion cysts called milia. Because the outer epidermal layer of the DEB patient adheres poorly to the underlying dermal connective tissue, even the slightest trauma will cause epidermal-dermal disadherence. Therefore, DEB patients suffer from chronic skin wounds. Studies have found that the poor skin adherence is due to a defect in the gene which encodes for type VII (anchoring fibril) collagen (C7); a protein that serves to anchor the epidermis onto the dermis [7,8].
At the molecular level, C7 is composed of three identical alpha chains, each consisting of a 145-kDa central collagenous triple-helical segment, flanked by a large 145-kDa amino-terminal, non-collagenous domain (NC1), and a small 34-kDa carboxyl-terminal non-collagenous domain (NC2) [9,10]. Within the extracellular space, C7 molecules form antiparallel dimers which aggregate laterally to form anchoring fibrils. In normal skin, C7 forms anchoring fibrils ranging from about 200-700 nm in size that emanate from epidermal-dermal junction (EDJ) and extend perpendicularly down into the papillary dermis. In DEB patients, the EDJ is characterized by a paucity of normal anchoring fibrils. Based on the underlying etiology of the disease, one logical approach for treating the disease is to correct the genetic defect through gene therapy.
There are several recent studies related to ex vivo gene therapy for DEB. In the study by Oritz-Urda et at., COL7A1 cDNA was successfully and stably integrated into C7-null keratinocytes from recessive DEB (RDEB) patients using a phi C31 integrase-based non-viral gene transfer approach. By transplanting a human skin equivalent comprising these gene-corrected cells onto severe combined immunodeficient (SCID) mice, they showed that many of the RDEB features were corrected after gene transfer
In another study by the inventors of the present invention, a minimal lentiviral vector was developed to express C7 in RDEB keratinocytes and fibroblasts (in which C7 was absent). This construct was subsequently used to demonstrate the reversion of the RDEB cellular phenotype [12]. In this experiment, the gene-corrected RDEB cells and native un-corrected RDEB cells were used to create a composite human skin equivalent which was then transplanted onto SCID mice. It was shown that the transplanted human skin made with the gene-corrected RDEB cells (but not the control un-corrected RDEB cells) exhibited C7 at the EDJ and the RDEB skin phenotype was corrected. Moreover, in the skin equivalents composed of gene-corrected (but not gene-uncorrected) cells, the transgene-derived C7 also created anchoring fibril structures that were correctly organized into the basement membrane zone (BMZ) lying between the epidermis and dermis.
However, this type of ex vivo approach requires transplantation of gene-corrected cells onto surgically prepared sites of the patient's skin. The experience of using cultured keratinocyte autografts for transplantation onto human wounds had shown that this technology is often fraught with technical difficulties and poor graft take. Therefore, although this ex vivo type of therapy (i.e. gene correcting cells in culture and then transplanting them back as skin equivalents onto the DEB patient) is theoretically possible, the technical hurdles make it in-efficient, logistically difficult, expensive, labor-intensive and of limited efficacy.
As an alternative approach, a more straightforward “direct in vivo gene therapy” was developed. With this approach, DEB wounded skin is directly injected through intradermal injection with gene-corrected RDEB fibroblasts. The gene-corrected intradermally injected cells then set up residence in the DEB skin and synthesize and secrete C7 which is lacking in the DEB skin. Surprisingly, the secreted C7 in the extracellular dermal tissue, binds to the BMZ of the DEB skin and correctly organizes into anchoring fibril structures. Now, the DEB skin which previously lacked C7 and anchoring fibrils, now has these elements and the DEB skin phenotype is corrected. The poor epidermal-dermal adherence is now corrected. This is called “cell therapy” for DEB by the inventors. The inventors also showed that the same events would occur if they intradermally injected full-length or “mini-C7” into DEB skin, the injected C7 would bind to the BMZ of the DEB skin and form correctly-organized anchoring fibrils and correct the DEB skin phenotype. The inventors called this “protein therapy” for DEB.
Full-length C7 contains a 39 amino acid interruption in the helical sequence which forms site that is highly susceptible to degradation by protease. The mini-C7 was designed by the inventors to contain the intact noncollagenous domains, NC1 and NC2, and half of the central collagenous domain. By excluding the 39 amino acid interrupt, this mini-C7 is made highly stable. The enhanced stability of mini-C7 over native C7 may better withstand proteolytic digestion in RDEB wounds and provide a more sustained gene product in patients. The inventors have shown that the mini-C7 is highly resistant to proteolytic digestion and yet when injected into DEB skin behaves identically to the full-length C7 in that it will bind to the BMZ, create new anchoring fibril structures and correct the DEB skin phenotype. Thirdly, the inventors showed that rather than intradermally injecting RDEB gene-corrected cells or C7 itself into DEB skin, they could inject simply into the DEB skin the lentiviral vector expressing either the full-length C7 or mini-C7. In this case, the exogenously injected vector was taken up by the endogenous dermal fibroblasts within the dermis of the DEB skin. These endogenous fibroblasts which previously lacked the ability to make C7 or mini-C7, now could synthesize and secrete these large proteins into the dermal extracellular environment. There, the C7 or mini-C7 homed to the BMZ of the DEB skin, organized into anchoring fibril structures and corrected the DEB skin phenotype. The inventors called this “vector therapy” for DEB [13-16, the relevant portions thereof are incorporated herein by reference].
In recent experiments, the inventors have shown that the intradermal approach is highly efficient if the agents are delivered in the high papillary dermis using a 30 guage needle with the bevel oriented upward, using a volume between 2 microliters and 2 milliters and injected into four quadrants of DEB skin between 1×1 cm and 6×6 cm.
In a more recent study, the investigators evaluated the feasibility of protein therapy in a C7 null DEB mouse which recapitulates the clinical and ultrastructural features of human RDEB. The investigators intradermally injected purified human C7 (into the new born DEB mice with severe skin blistering and fragility and found that the injected human C7 transported and stably incorporated into the mouse's BMZ and formed anchoring fibrils. The restoration of C7 corrected the DEB murine phenotype as demonstrated by decreased skin fragility and blistering, reduced new blister formation and marked prolonged survival.
Despite the above mentioned advances, there are still no effective methods for treating DEB. Because patients with severe DEB have widespread lesions and multiple wounds spanning large areas of trauma-prone sites such as the sacrum, hips, feet, mouth, scalp, lower back and hands, the treatment of such DEB patients via any of the three above outlined direct intradermal injection approaches would require numerous injections into multiple wound sites. Accordingly, intradermal injections of the therapeutic agents outlined above (gene-corrected cells, recombinant forms of C7 or C7 expressing vectors) would require site-specific treatment of each and every wound by one or more intradermal injections. While this is doable, such a cumbersome method of treatment still leaves much to be desired. It would be ideal to offer patients with DEB or patients with multiple wounds a single therapy that will require only a single route of delivery but that will “home” to all of the wounds automatically upon delivery to correct the skin wounds located at scattered sites.
Therefore, there still exists a great need for better method of treating skin wounds in general and DEB in particular.